Maximizing benefits from ‘biodiscovery’: a Coastal State ...€¦ · treatment for treatment for inflammatory diseases New compound ‘Stylissadines A and B’specific antagonist

Maximizing benefits from ‘biodiscovery’: a Coastal State resource providers perspective

John N.A. Hooper, Queensland Museum & Natural Products Discovery Griffith University, Brisbane

Research State (user of the marine genetic resources (MGRs) for biodiscovery purposes)

Coastal State (provider of MGRs to a Research State)

Biodiscovery (= bioprospecting, and in this instance, refers only to marine natural products discovery)

Some definitions …

An Australian perspective from a Queensland experience

• Enabling environment: How does a Coastal State attract a Research State to use their MGRs ?

o the ‘stick’: regulatory protocols, policy & legislation

o the ‘carrot’: access to MGRs in Australia & Queensland

• Experiences in major biodiscovery collections

• Research State benefits: an example of a successful biodiscovery partnership

• Coastal State benefits: ‘environmental data’ from collections for bioregional planning, new biological, genetic and chemical data, taxonomic capacity building, etc

The (dis)enabling biodiscovery environment: •Myriad of Laws governing biodiscovery: E.g.

o Forestry Acto Land Acto Nature Conservation Acto Fisheries Acto (State) Marine Parks Acto Great Barrier Reef Marine Park Acto Native Title Acto Wet Tropics World Heritage Protection and Management Act o Etc.

‘Biodiscovery’ regulatory framework pre-2004

‘Biodiscovery’ regulatory framework pre-2004The (dis)enabling biodiscovery environment: Myriad of Laws:• Many access permits from many agencies: E.g. o Marine Parks collections Permit, The Great Barrier Reef Marine Park Authority Regulations 1983 (Commonwealth)o EPA Collections Permit, Environmental Protection Agency, Queensland Parks & Wildlife Service, Marine Parks Regulations 1990 (Queensland)o DPI General Fisheries collections Permit, Department of Primary Industries and Fisheries, Queensland Fisheries Act, 1994o Museum Licence, Environmental Protection Agency, Queensland Parks & Wildlife Service, s170f Nature Conservation Regulation 1994.o Permit to ‘Take, Use, Keep or Interfere with Cultural or Natural Resources (Scientific Purpose)’,Environmental Protection Agency, Queensland Parks & Wildlife Service, S9(1)(A) Nature Conservation (Administration) Regulation 2006.o Commercial wildlife harvest licence under the Nature Conservation Act 1992o DNR Sales permit under the Forestry Act 1959 to remove materialo Commercial wildlife licence under the Nature Conservation Act 1992 to use the material.

‘Biodiscovery’ regulatory framework pre-2004The (dis)enabling biodiscovery environment: Myriad of Laws: • Many access permits from many agencies:• Lack of clarity:

o ownership of MGRso right of access

o rights to commercialisation, IP, etc from use of MGRs

‘Biodiscovery’ regulatory framework pre-2004The (dis)enabling biodiscovery environment: Myriad of Laws: • Many access permits from many agencies:• Lack of clarity:• Potentially ecologically unsound and unsustainable collection activities:

o few, if any, environmental benefitso research effort uncoordinatedo new knowledge not in public domain

‘Biodiscovery’ regulatory framework pre-2004The (dis)enabling biodiscovery environment:Myriad of Laws: • Many access permits from many agencies:• Lack of clarity:• Potentially ecologically unsound and unsustainable collection activities:• Little or no investment in Coastal State R&D:

o mostly offshore commercialisation & value-addingo little or no investment in infrastructure, capacity building, compound

librarieso no contribution to Coastal State voucher specimen collections

(environmental knowledge)

‘Biodiscovery’ regulatory framework pre-20042002: Bonn Guidelines adopted by the CBD to

provided extra guidance on ABS2002: Nationally Consistent Approach agreed by all

states, territories and Commonwealth of Australia

2004: Queensland 1st state to implement nationally consistent approach (CBD + Bonn Guidelines) = ‘Queensland Biodiscovery Act 2004’

Post 2004: 2 other Australian jurisdictions have new legislation; 4 others currently developing policy

• (http://www.legislation.qld.gov.au/LEGISLTN/ACTS/2004/04AC019.pdf)

The enabling environment: Purpose of the Act: Purpose of the Act: 1. Regulatory: provide certainty for all stakeholders by establishing

a streamlined and clear legislative framework to regulate collection and use of all Queensland’s native biological materials from lands and waters;

The ‘stick’: Queensland Biodiscovery Act 2004

Tools:Biological Collection Authority (BCA):• single permit issued minimising duplication of

regulation; • regulated by a set of Codes (ethics,

collection standards, minimising environmental damage etc)


The enabling environment: Purpose of the Act:Purpose of the Act:2. 2. Co-investment: encourage investment in the Queensland’s

biodiscovery research and development sector;3. 3. Commercialisation: ensure fair and equitable benefit sharing

from biodiscovery to Queensland;


Tools:Benefit Sharing Agreement (BSA):i.e. royalties, investment into Queensland's biotechnology industry, collaboration with researchers, development of new or improved products, obligation to lodge voucher samples in museums/ herbaria


The enabling environment: Purpose of the Act:Purpose of the Act:4. 4. Environmental: enhance knowledge of Queensland’s biodiversity,

promoting conservation and ecologically sustainable use of native biological resources.


Tools:Biodiscovery Plan:developed by Research State in line with the Codes (e.g. source and scope of biological material, collection access, timetable & localities, proposed commercial outcomes, environmental data and other non-monetary benefits etc.)


The enabling environment: Purpose of the Act:Purpose of the Act:4. 4. Environmental: enhance knowledge of Queensland’s biodiversity,

promoting conservation and ecologically sustainable use of native biological resources.


Non-monetary benefits:‘environmental data’ arguably as valuable as any potential monetary benefits from commercialisation.

• BigBig (70,000km continental coastline, 8.6 million km2 continental marine territory; 16.1 million km2 oceanic jurisdiction);

• DiverseDiverse (60 marine bioregions, all 5 oceanic climatic zones, tropics to polar, intertidal to the abyssal plains)

• Highly UniqueHighly Unique (1 of the 19 ‘megadiverse’ countries, huge proportion of endemic biota,Gondwanan origins & subsequent mixing with the Tethyan biota)

The ‘carrot’: Australian Marine Genetic Resources (MGRs)

17 of Australia’s 60

marine bioregions

5 of Australia’s 13

world heritage-listed sites (including

the Great Barrier Reef WHA, Great

Sandy Region –Fraser Island)

GBR Qld Plateau

emergent reefs = Coral Sea territories

NE seamounts

Gulf Carpentaria

Great Sandy Region

The ‘carrot’: Queensland MGRs

Sponges: significant scientific progress over past 2 decades, and most number of new compounds discovered from biodiscovery

(Data from US (Data from US National Cancer National Cancer Institute; Institute; Munro et al., Munro et al., 1999)1999)

Bryozoans

AscidiansSoft corals, gorgonians

Sponges

Toxicity at low ‘tissue’ concentration

Major marine target animals for biodiscovery

• 1974-1981: Roche Research Institute of Marine Pharmacology & Roche Pharmaceuticals• 1987-1992; 2003-present: Australian Institute of Marine Science (AIMS) & US National Cancer Institute (NCI)• 1993-present: Natural Products Discovery Griffith University (NPD) (+ Queensland Museum, Queensland Herbarium) & AstraZeneca (AZ)

Experiences in major ‘Biodiscovery’ collections (PPP)10kg biomass samples

collected 6 extracts screened/

week~2,100 extracts

screened over 7 years

100g biomass samples collected (200mg extract for screening)

180,000 extracts screened per dayModel example

• Cardiovascular• Respiratory • Inflammation• Gastrointestinal• Pain Control• Oncology• Infection• CNS

Specimen library(>30,000 plants, animals, microorganisms)

20,029

7,554

1,207

6,364 5,673

1,507 956

0

5,000

10,000

15,000

20,000

25,000

Number of BiotaQueensland Plants Queensland Marine OrganismsMicrobials China PlantsPNG Plants Queensland Terrestrial OrganismsTasmanian Marine Organisms

NPD/AZ partnership: Research State NPD/AZ partnership: Research State ‘‘commercialcommercial’’ benefitsbenefits

Extract Library(2D dot matrix coded microtube; 5 year shelf life; 384-well - 50 mL assay volume; 200 mg of sample gives > 800 assays)


NPD/ AZ partnershipNPD/ AZ partnership

High throughput screening:focussing on: Receptors, Enzymes, Mechanism based cellular assays(Cardiovascular, Respiratory, Inflammation, Gastrointestinal, Pain Control, Oncology, Infection, CNS)



Biota extract

Automated liquid handling and Screening

Fraction collection

ChromatographyRP / NP / Ion Exchange

Gel PermeationCountercurrent

AutomatedSeparation

Pure CompoundStructure Determination

MS / NMR Analysis

CRC

mixture

NHN

NH

HN

HN

O

HN

O

H2N

H2N

NH

NH

Br

Br

Br

Br

OH H

H ClH

OH

• Bioassay guided

fractionation• Isolation of

active constituent• Structure elucidation

• Lead optimisation



•Potential accessible chemical diversity (compounds with >0.001% yield) = 5-7 million compounds

~1500 bioactive compounds isolated to date, 23 new structure classes discovered, 38% are novel structures, majority with novel biological activity

Known Compound-

new activity

57%

Known Compound-

known Activity

5%

New Compound

38%

1

AU

0 . 0 0

0 . 5 0

1 . 0 0

1 . 5 0

2 . 0 0

2 . 5 0

3 . 0 0

M in u t e s2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0 3 0 . 0 0

1% yield

0.1% yield 10

10010000.01% yield

Compounds/biota


O

HN NH

NHHN

H2N

NH

HN Br

Br

O

HN

O NH

Br

Br

OHH

O

NHHN

HN NH

NH2

HN

NHBr

Br

O

NH

OHNBr

Br

HOH

O

4CF3COO

NH2

NH2

Stylissa flabellata from the GBR

P2XP2X77 antagonists may provide new antagonists may provide new treatment for treatment for inflammatory diseasesinflammatory diseases

New compound ‘‘StylissadinesStylissadines A A and Band B’’ specific antagonist for P2XP2X77(pain) receptor - purinergic ligand-gated ion channel (non-cytotoxic, IC50 1.8 mM)

(Buchanan et al. J. Org. Chem., 2007, 72, 2309-2317)


O

HN NH

NHHN

H2N

NH

HN Br

Br

O

HN

O NH

Br

Br

OHH

O

NHHN

HN NH

NH2

HN

NHBr

Br

O

NH

OHNBr

Br

HOH

O

4CF3COO

NH2

NH2

Stylissa flabellata from the GBR

P2XP2X77 antagonists may provide new antagonists may provide new treatment for treatment for inflammatory diseasesinflammatory diseases

New compound ‘‘StylissadinesStylissadines A A and Band B’’ specific antagonist for P2XP2X7 7 (pain) receptor - purinergic ligand-gated ion channel (non-cytotoxic, IC50 1.8 mM)

CSIRO CAAB bioclim model

(Buchanan et al. J. Org. Chem., 2007, 72, 2309-2317)

Slope and sides of coral reefs, below 10m depth


Aplysinella rhaxfrom the GBR

(Pham et al, J. Nat. Prod. 2000, 63, 393 – 395)

Modulates Modulates cAMPcAMP via adenylate via adenylate cyclase effective for controlling cyclase effective for controlling blood flowblood flow, , cardiovascular systemcardiovascular systemand and metabolismmetabolism

Adenosine A1 antagonists PsammaplinPsammaplin AAand BisaprasinBisaprasin 1111’’--sulfatesulfate [3H]DPCPX binding to brain adenosine A1 receptors IC50

OHBr

NH

S SNHN

O O

NHO OH

ORBr

OHBr

NH

S SNHN

O O

N

HN

SSHN

OSO3NaBr

Br

OHOH

Br

N

OO

N

HOHO

OHOH


Aplysinella rhaxfrom the GBR

(Pham et al, J. Nat. Prod. 2000, 63, 393 – 395)

Modulates Modulates cAMPcAMP via adenylate via adenylate cyclase effective for controlling cyclase effective for controlling blood flowblood flow, , cardiovascular systemcardiovascular systemand and metabolismmetabolism

Adenosine A1 antagonists PsammaplinPsammaplin AAand BisaprasinBisaprasin 1111’’--sulfatesulfate [3H]DPCPX binding to brain adenosine A1 receptors IC50

OHBr

NH

S SNHN

O O

NHO OH

ORBr

OHBr

NH

S SNHN

O O

N

HN

SSHN

OSO3NaBr

Br

OHOH

Br

N

OO

N

HOHO

OHOH

CSIRO CAAB bioclim modelCoral rubble, rock, shallow water


Adocia (=Haliclona) aculeata from the GBR

(Kalaitzis et al, J. Org. Chem. 1999, 64, 5571 – 5574)

OH

H H

-O3SO

OH

HO

AdociasulfateAdociasulfate 1,7,8,91,7,8,9iinhibits H+-ATPase proton pump IC50 essential to osteoclast bone resorption cell activity

HOH

H H

RO

OSO3-

OH

H H

H

-O3SO

H

OSO3- AdociasulfateAdociasulfate may reduce the may reduce the

rate of bone resorption and offer rate of bone resorption and offer therapeutic value in therapeutic value in osteoporosisosteoporosis



Adocia (=Haliclona) aculeata from the GBR

(Kalaitzis et al, J. Org. Chem. 1999, 64, 5571 – 5574)

OH

H H

-O3SO

OH

HO

AdociasulfateAdociasulfate 1,7,8,91,7,8,9iinhibits H+-ATPase proton pump IC50 essential to osteoclast bone resorption cell activity

HOH

H H

RO

OSO3-

OH

H H

H

-O3SO

H

OSO3- AdociasulfateAdociasulfate may reduce the may reduce the

rate of bone resorption and offer rate of bone resorption and offer therapeutic value in therapeutic value in osteoporosisosteoporosis

CSIRO CAAB bioclim modelencrusting on corals and gorgonians, deeper lagoon >15m depth


New species of marine sponge Citronia astra

from outer GBR

N

HN

HOH

H

O

HO

N NH2

NH2

NH

O

O

OSO3H3CO

New compound ‘‘DysinosinDysinosin AA’’,found to inhibit the binding of [I125] interleukin-8 [IL-8] to the human recombinant IL-8 receptor type A at IC50 = 154 nM

(Leone et al, J. Nat. Prod. 2000, 63, 694 -697)

‘‘DysinosinDysinosin AA’’ found to be effective found to be effective in in oncologyoncology, awaiting Phase I trials, , awaiting Phase I trials, and also another compound effective and also another compound effective against human against human aa--thrombinthrombin


New species of marine sponge Citronia astra

from outer GBR

N

HN

HOH

H

O

HO

N NH2

NH2

NH

O

O

OSO3H3CO

New compound ‘‘DysinosinDysinosin AA’’,found to inhibit the binding of [I125] interleukin-8 [IL-8] to the human recombinant IL-8 receptor type A at IC50 = 154 nM

(Leone et al, J. Nat. Prod. 2000, 63, 694 -697)

‘‘DysinosinDysinosin AA’’ found to be effective found to be effective in in oncologyoncology, awaiting Phase I trials, , awaiting Phase I trials, and also another compound effective and also another compound effective against human against human aa--thrombinthrombin

CSIRO CAAB bioclim modelOuter barrier reef flat, on coral pavement, <5m depth


New species of Axinellasp.1333 from Sydney

AxinellaminesAxinellamines B, C and DB, C and D: minimum bactericidal concentration for inhibition of H. pylori of 1000 mM

NHN

NH

HN

HN

O

HN

O

H2N

H2N

NH

NH

Br

Br

Br

Br

OH H

H

OHHCl

NHN

NH

HN

HN

O

HN

O

H2N

H2N

NH

NH

Br

Br

Br

Br

OH H

H

OHHCl

(Urban et al, J. Org. Chem. 1999, 64, 731 – 735)

Gastric bactericideGastric bactericide potentially potentially effective for treatment and effective for treatment and prevention of gastric and duodenal prevention of gastric and duodenal ulcer and gastric cancerulcer and gastric cancer


New species of Axinellasp.1333 from Sydney

AxinellaminesAxinellamines B, C and DB, C and D: minimum bactericidal concentration for inhibition of H. pylori of 1000 mM

NHN

NH

HN

HN

O

HN

O

H2N

H2N

NH

NH

Br

Br

Br

Br

OH H

H

OHHCl

NHN

NH

HN

HN

O

HN

O

H2N

H2N

NH

NH

Br

Br

Br

Br

OH H

H

OHHCl

(Urban et al, J. Org. Chem. 1999, 64, 731 – 735)

Gastric bactericideGastric bactericide potentially potentially effective for treatment and effective for treatment and prevention of gastric and duodenal prevention of gastric and duodenal ulcer and gastric cancerulcer and gastric cancer


Common on rocky substrates, 8-35m depth, but Sydney region only


QM sponge collections: ~4,000 collection sites, ~30,000 specimen records,~5,000 morphospecies; datapoints all underpinned by verifiable objects

Coastal State benefits: environmental data (collections)

NPD/ AZIRD Noumea / CRISP

Univ Utah/ UPNG/ Wyeth/ ICBG - NIH

AIMS/ US NCI

Capacity building: Capacity building: Taxonomy (theory and practice of classifying organisms; systematics, biogeography)

Collections: verifiable objects that underpin biodiversity and biodiscovery research

Taxonomy: enabling science underpinning most other biological sciences;

essential to reliably differentiate MGRs in an international context (not to be confused with ‘sorting’ or ‘identification’)

Taxonomic capability: in crisis (Australia) 1980s: ~ 190 full-time (long-term employed) taxonomists2006: ~ 75 full-time taxonomists in museums, herbaria,

universities, other government agencies

Capacity building: Capacity building: Taxonomy (GBR Seabed biodiversity)•150,000 specimens collected over 3 years throughout GBR inter-reef•most identified to OTU level (i.e. same vs. different)•but few yet identified to species (many new ?)•Without true taxonomy, how can we determine if species are unique to the GBR or found in other Coastal States ?

unique or ubiquitous ?

SPICULES

Astrosclera`willeyana´

Morphological (phenotype) versus genotype species concept problem (ultraconservative sponge morphologies, deeply divergent genetically)(data Wörheide, 1998, Facies, 38: 1-88)

~1,500~500

Unknowable Unknowable (too (too difficult to difficult to collect, collect, DNA DNA probes)probes)

~15-17,000

~7,000~8,000Worldwide

~5,500-6,000

~2,000~3,500

~1,200~1,600

Australia (1992)(2006)

TotalsTotalsUnknown Unknown (collected, (collected, not not described)described)

Known Known (described)(described)

Capacity building: Capacity building: Taxonomic inventory

Coastal State benefits: Species diversity & similarity

10%10%

25%25%21%21%

8%8%

15%15%

18%18%

Tweed River (69)

Regional faunas

SE. Qld(233)

QldPlateau (95)

GBR (507)

Gulf of Carpentaria (142)

Sydney (131)

( = 50 spp)

(Hooper et al. Mem. Qld. Museum, 1999, 44: 263-274)

Biodiversity ‘hotspots’ (species rich, >250 spp/ region)

Species Species richnessrichness

(Hooper, Kennedy & Quinn, Biodiv. & Conserv., 2002, 11(5): 851-885)

Coastal State benefits: ‘Hotspots’ of species richness

Species poor regions (<50 spp/ region)

• Taxonomic relationships

• Evolutionary/ biogeographic history

• Artefacts of ancient connectivity

• Influenced by modern physical attributes (currents, sediments, etc)

Coastal State benefits:Phylogeneticrelationships

(Hooper, Kennedy & Quinn, Biodiv. & Conserv., 2002, 11(5): 851-885)

Coastal State benefits: molecular studies, genetic diversity versus morphological diversity (connectivity, isolation, hybridization: Leucetta `chagosensis´)

(Wörheide, Hooper & Degnan, Molecular Ecology , 2002, 11: 1753-1768)

ITS 1&2 + partial 28S (C2D2), 875+372bp; N=127

Genetic distinctiveness (Leucetta chagosensis)

S-Pacific

S-GBR, PNG, + QP

N-GBR + N-Pacific, QPMrBayes: ITS 1&2 + partial 28S (C2D2)

875+372bp; N=127

IndonesiaMaldives, Vanuatu (1)


!!! Deep genetic divergence of N. & S. GBR populations

Genetic clade 3-1 southern GBR to Brisbane

Genetic clade 3-5 northern/ central GBR

Genetic clade 3-2 S-Pacific

Inferences from molecular data: Deep divergence in GBR


Pleistocene Hypothesis:recolonization of the Great Barrier Reef after the low sea levels during the Pleistocene (1.8 M –12 K yrs ago)


Modern day

Species refuges during destruction of GBR in Pleistocene

SEC


Hypothesis:recolonization of the Great Barrier Reef after the low sea levels during the Pleistocene (1.8m –12k yrs ago)

Over the last 12,000 yrs

Exogenous recruitment

Reconnected populations (hybridization)

SEC

Climate change response ? Change of sea currents, new patterns of connectivity, increased severe storm events ? = loss of native faunas, increased invasive species via storm events (‘blowins’) ?

The future ?

?Today: marginal coral reef ecosystems

Tomorrow: important refuges for recruitment ?

Primers The standard barcoding fragment for the Barcoding of Life initiative is a ~ 640 bp long fragment located at the 5' site of the mitochondrial cytochrome oxidase subunit 1 (CO1). However, as this fragment might not display sufficient variability in sponges [1][2], we suggest sequencing an additional downstream region, which exhibits a higher substitution rate [3] in order to obtain a fragment of about 1,100 bp with a higher number of variable sites. A submission ITS-1 and 2 sequences, which could be added to the CO1-sequence tag, is also encouraged.

~600 sequences contributed to sponge barcodingproject (CO1, ITS-1 and 2) from samples collected from NPD/ AZ bio-discovery

http://www.spongebarcoding.org/

Qld species from biodiscovery used for sponge genome project

Coastal State benefits: Coastal State benefits: ‘‘Species turnoverSpecies turnover’’ –– Bioregional Bioregional boundaries boundaries E.g. E.g. CymbastelaCymbastela speciesspecies

Physical barrier or environmental gradient (such as during the Pleistocene landbridge)

Lack of gene flow across barrier

Coastal State benefits: Coastal State benefits: ‘‘Species turnoverSpecies turnover’’ –– Bioregional Bioregional boundaries boundaries (e.g.e.g. EchinodictyumEchinodictyum species) species)

Coastal State benefits:Coastal State benefits: ‘‘Species turnoverSpecies turnover’’, Bioregional , Bioregional modellingmodelling (aggregated data for ~3,500 species)(aggregated data for ~3,500 species)(Hooper & Ekins, 2004, National Oceans Office C2004/020: Hobart, http://www.oceans.gov.au/NMB.jsp).

Theoretical IMCRA DEMERSAL BIOREGIONS: 1999

Coastal State benefits:Coastal State benefits: ‘‘Species turnoverSpecies turnover’’, Bioregional , Bioregional modellingmodelling (aggregated data for ~3,500 species)(aggregated data for ~3,500 species)(Hooper & Ekins, 2004, National Oceans Office C2004/020: Hobart, http://www.oceans.gov.au/NMB.jsp).

Theoretical IMCRA DEMERSAL BIOREGIONS: 1999

Coastal State benefits:

Great Barrier Reef Marine Park Authority Representative Areas Program

Summary:

Research State benefits: legislative certainty for access, IP, benefit sharing etc.; partnerships with Coastal State researchers infrastructure; biodiscoveryproducts including building extract and compound libraries; discovery of new compounds and bioactive leads; potential commercialization = $ …

Coastal State benefits: ‘environmental data’ from biological collections, genetic and biochemical research; taxonomic and other capacity building support; contribution to barcoding projects; new knowledge pivotal to bioregional marine planning, management and ultimately conservation of MGRs; potential royalties …

Documents

Maximizing benefits from ‘biodiscovery’: a Coastal State ...€¦ · treatment for treatment for inflammatory diseases New compound ‘Stylissadines A and B’specific antagonist